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Abstract

Background

Despite the fact that the implication of human papillomavirus (HPV) in the carcinogenesis
and prognosis of cervical cancer is well established, the impact of a co-infection
with high risk HPV (HR-HPV) and Epstein-Barr virus (EBV) is still not fully understood.

Methods

Fifty eight randomly selected cases of squamous cell carcinomas (SCC) of the uterine
cervix, 14 normal cervices specimens, 21 CIN-2/3 and 16 CIN-1 cases were examined
for EBV and HPV infections. Detection of HR-HPV specific sequences was carried out
by PCR amplification using consensus primers of Manos and by Digene Hybrid Capture.
The presence of EBV was revealed by amplifying a 660 bp specific EBV sequence of BALF1.
mRNA expression of LMP-1 in one hand and protein levels of BARF-1, LMP-1 and EBNA-1
in the other hand were assessed by RT-PCR and immunoblotting and/or immunohischemistry
respectively.

Results

HR-HPV infection was found in patients with SCC (88%), low-grade (75%) and high grade
(95%) lesions compared to only 14% of normal cervix cases. However, 69%, 12.5%, 38.1%,
and 14% of SCC, CIN-1, CIN-2/3 and normal cervix tissues, respectively, were EBV infected.
The highest co-infection (HR-HPV and EBV) was found in squamous cell carcinoma cases
(67%). The latter cases showed 27% and 29% expression of EBV BARF-1 and LMP-1 oncogenes
respectively.

Conclusion

The high rate of HR-HPV and EBV co-infection in SCC suggests that EBV infection is
incriminated in cervical cancer progression. This could be taken into account as bad
prognosis in this type of cancer. However, the mode of action in dual infection in
cervical oncogenesis needs further investigation.

Keywords:

Background

Cervical cancer is the second most prevalent cancer among the Algerian women. The
association between human papilomavirus (HPV) and cervical neoplasia is well documented
[1]. High risk oncogenic HPV types (including HPV 16 and HPV 18) are associated with
99.7% of all low-grade cervical (CIN-1 or mild CIN) and high grade intraepithelial
lesions (CIN-2/3) and hence, they play an important role in cervical cancer development.
Now, and since 1976, it is well recognized that HPV infections in the cervix are frequently
associated with intraepithelial neoplasia and invasive squamous cell carcinomas (SCC)
with all their different histological variants (large-cell keratinizing, large-cell
non-keratinizing and small-cell carcinoma).

The long period of time (years) it takes for the development of cervical cancer after
HPV infection suggests the involvement of other etiologies (such as viruses or cell
compounds) in malignancy process. The synergistic effect of carcinogenic factors such
as two or more viruses interacting at different stages of tumor development has been
reported [2-4]. Epstein-Barr virus (EBV), ubiquitous human gamma-herpes virus responsible for mononucleosis
[5], could be one of the ‘helper’ viruses. It can be sexually transmitted [5] and replicates in cervix cells [6]. EBV infection, widely spread among the population [7,8], has been associated with an increasing number of lymphocytic and epithelial cancers,
mainly Burkitt’s lymphoma, Hodgkin’s lymphoma, T cell lymphoma, nasopharyngeal carcinoma
(NPC) and gastric adenocarcinoma [9,10].

BARF1 is one of the EBV-encoded proteins secreted in the serum of NPC patients [11] and expressed in more than 90% of NPC biopsies [12-15] and tumor epithelial cells of EBV-associated gastric carcinoma [12]. It has a malignant transforming activity in rodent fibroblasts [16] and in EBV-negative human B cells [14]. LMP1, another EBV oncogene candidate essential for B cell immortalization [17], was present in 30 to 50% of NPC biopsies [18]. This oncogene can activate a number of cellular key genes such as NFκB and EGFR
[17,19]. LMP-1 can inhibit cell differentiation when transfected into epithelial cells [18].

Tseng et al. [20] reported a high incidence of EBV in lymphoepithelial like-carcinoma (LELC) patients
but did not show any association with HPV. These findings are in contradiction with
what has been previously reported [21,22]. Therefore, the oncogenic relationship between the two viruses remains not fully
understood. Added to this, the presence of EBV in the cervix carcinoma remains equally
a topic of great debate among virologists, confirmed by certain authors [2,23,24] but not by others [25,26].

As it is well known EBV can transform cells bearing the EBV/C3d receptor making them
receptive to other oncogenic stimuli [27]. These receptors are widely detected on ecto- and endo-cervical biopsies of the uterine
cervix [28-30]. EBV replicates in cervical epithelium and its possible role in cervical carcinoma
development has been raised.

We looked, in this study, for the presence of both EBV and HPV DNA sequences in Algerian
patients with SCC and cervical lesions. We examined the presence of EBV infection
and the EBV-HPV co-infection. The presence of EBV in cervical cancer tissues suggests
its possible involvement in the cervical cancer progression. Initially, PCR amplification
was used to identify the co-infection. This was followed by an investigation on EBV
oncogenes (BARF-1, LMP-1 and EBNA-1) expression using immunoblotting and immunohistochemistry.
This expression would reflect the transformation mechanism of cervical cells.

Since EBV could play an important role in Nasopharyngeal carcinoma and Burkitt’s lymphoma
highly frequent in Algeria, Our hypothesis was a possible co-infection by HPV and
EBV in Algerian SCC and cervical lesions.

Results

Detection of HR-HPV and EBV in SCC biopsies

Fifty eight biopsies of SCC from Algerian women were analyzed to assess the presence
of low and high-risk HPV strains. PCR and Hybrid Capture 2 (HC2) tests revealed that
out the 58 studied samples 51 (88%) were positive to HR-HPV strains and 7 (12%) were
HPV infection free. Among these HPV cases, 40 biopsies were exclusively infected by
high-risk HPV strains and 11 were co-infected with high- and low-risk HPV (Figure 1).

In the same biopsies, the EBV genome was detected by PCR amplification of the entire
EBV-encoded BALF1 gene. The PCR product (BALF1 sequence of 660 bp) was confirmed by
Southern blot hybridization using the random primer kit with 32P-labeled BamH1-A fragment as a probe (Figure 2). As illustrated in Figure 1, 69% (40/58) cases of SCC cervical biopsies were positive to BALF1 sequence. EBV
BALF-1 was present in only one case that was HPV negative. 67.2% (39/58) were co-infected
with both viruses. 20.7% (12/58) were only HPV infected. Co-infections with HPV and
EBV were detected more frequently in CIN-2/3 lesions and SCC groups than in NC and
CIN-1 groups (p < 0.05). In terms of co-infection, there was no significant difference
between NC and CIN-1 groups.

Figure 2.Detection of EBV BALF1 gene by PCR. Amplified PCR products were electrophoresed and the presence of BALF1 sequence [39] was confirmed by hybridization with 32P labeled BamH1-A fragment used as a probe. A: lanes 1-8 for tumor samples and B: lanes 9-16 for precancerous lesions. B95-8 DNA and H2O were used as positive and negative controls respectively.

PCR detection of HR-HPV and EBV in cervical lesions and in normal patients

Using PCR and HC2, 21 CIN-2/3 and 16 CIN-1 lesions and 14 normal tissue samples were
investigated for HPV genome. The two methods showed a good correlation. 95% (20/21)
CIN-2/3 cases were infected by HR-HPV compared to 40% (8/21) were co-infected with
EBV. Among the 16 CIN-1 cases, HR-HPV was present in 75% (12/16) with 12.5% (2/16)
of co-infections. The remaining twenty three EBV free cases (CIN-1 and CIN-2/3) were
infected with either high or/and low risk HPV strains. Among the fourteen normal cervix
samples, only two were HR-HPV positive and only one was co-infected (Figure 1).

Transcriptional and translational expression of EBV in SCC cases

Limited by tissue specimens, the mRNA (LMP-1) and protein (BARF-1) expression of EBV
oncogenes was assessed in only 23 out of 58 samples. However, all the three proteins
(BARF-1, LMP-1 and EBNA-1) were immunohistochemically assessed in 45 out 58 of SCC
biopsies (Table 1).

Table 1.Relationship between the detection of EBV oncoproteins (LMP-1 and BARF-1) and the
presence of either or both HPV and EBV in malignant cervical biopsies

As shown in Figure 3, the LMP-1 transcript was about 200 bp which was spliced from the 406 bp DNA sequence
(see Material and Methods). Using immunoblotting technique, BARF-1 protein was detected
in only 21.7% (5/23) of SCC biopsies analyzed from 50 μg cellular extract (Figure 4). We also examined the transcriptional and translational expression of HPV and EBV
oncogenes (LMP-1 and BARF-1) by immunohistochemistry (IHC) (Figure 5). IHC analysis has allowed the identification of HPV and EBV products. As shown in
Figure 5D, HPV was expressed in SCC biopsies. EBV antigens, EBNA-1 and LMP-1, were present
as a brown nuclear and membrane staining (Figure 5B and C respectively). EBNA-1 protein was detected in 34.7% (8/23). While IHC analysis
showed 26% (6/23) LMP-1 positive cases (Figure 5C). Negative squamous carcinoma cells for EBNA-1 and LMP-1 proteins are shown in Figures 5E and F respectively.

Figure 4.Detection of BARF-1 protein by Western blotting. Immunoblotting of BARF-1 on seven cervical tumor biopsies. Fifty micrograms of protein
extract as measured by a Biorad protein assay were deposited onto 12% polyacrylamide
gel and electrophoresed. Proteins were transferred onto nitrocellulose paper. BARF1
protein encoding 29 kDa (p29) was revealed by PepIII rabbit polyclonal anti-BARF-1.
BARF-1 protein (p29) produced by the BARF-1 recombinant adenovirus system was used
as a positive control indicated in first lane as BARF1.

Discussion

Our findings point out that most patients with either SCC or cervical lesions are
HR-HPV infected. To our surprise, EBV-HPV co-infection was detected in the majority
(67%) of cervical carcinoma among the Algerian women involved in this study. The presence
of EBV was found in 69% of cervical cancer biopsies, suggesting its possible association
with the development of cervical cancer. This is in agreement with previous reports
[2,23,24]. However, other researchers, using in situ hybridization, reported controversial
results [31-33]. Furthermore, Sasagawa et al. [24] demonstrated, by RNA in situ hybridization, the presence of EBV and the expression
of EBV genes, EBER-1, LMP-1 and EBNA-1, in cervical biopsies.

Using immunohistochemistry technique, to our knowledge, we are the first to detect
EBV antigens in the cervix tissue. The difference in our results between the PCR method
and protein detection has three possible explanations; (i) DNA amplification using
PCR is a more sensitive technique than the detection of antigens using Immunohistochemistry
or immunoblotting, (ii) EBV genome is present but its genes are not abundantly expressed
in EBV-infected cells and (iii) a contamination by EBV-positive B lymphocytes infiltrating
the connective tissue or EBV-positive normal cervical epithelium adjacent to the lesions.
PCR results on their own merely indicate the presence of EBV in the cervix tissue
but not necessarily in the epithelial cells.

Our data suggest that co-infection with EBV and HR-HPV may be of cervical significance
in the ethiopathogenesis of uterine cervical cancer. However, few cells in cervical
tissue were infected by EBV and harbor EBV LMP-1 protein (Figure 5). The expression of LMP-1 and EBNA-1 in SCC may lead to the destruction of cancer
cells as they are targets for EBV-specific cytotoxic T lymphocytes. Such process has
been reported in gastric [12] and Burkitt lymphomas [10]. However, the cervix is histologically different from lymphoid tissues which are
localized very far from the cervical epithelium by thick myometrial tissue of the
cervix. The same situation has been observed in HPV infection where E6-E7 oncogenes
are frequently expressed in cervical neoplastic lesions and which are equally targets
for cytotoxic T lymphocyte response in patients with cervical neoplasia [34].

EBV DNA has been shown in the exfoliating cells [28]. The virus could infect cultured ectocervical cells and express its late antigens
suggesting an association between EBV replication and epithelial differentiation [35]. Infectious EBV able to transform B lymphocytes has been isolated from cervical washes
from women recovering from EBV mononucleosis infection or EBV-seropositive with no
acute infection. Several studies showed that EBV infection is sexually transmitted
targeting the uterine cervix [5,36-38].

In this study, EBV latent genes were expressed in some squamous epithelia. The cervix
might be a site for chronic EBV shedding in a similar manner to the nasopharynx. It
is widely accepted that EBV infects B lymphocytes through CD-21 (EBV/C3d) receptor
[27] which are equally present in cervix cells. The squamocolumnar junction of the cervix
is the area where tissue repair and inflammation frequently occur, and where cervical
malignancies associated with HPV infection develop. Similarly, chronic cervicitis
also may help the EBV infection [4]. The low rate of EBV infection in normal cervixes and in pre-cancer lesions compared
with SCC cases of patients used in this study suggests that EBV may play a role in
late cervical carcinogenesis. Further investigation is required to clarify whether
the presence of EBV is a worse prognosis for the development of cervical cancer. EBV
may be acting as a cofactor with another carcinogenic agent(s), possibly human papillomaviruses,
in the final invasive cancer progression.

Conclusion

Preliminary presented results of EBV and HR-HPV co-infection in Irish, North American,
Thailand and Japanese SCC cases were recently reported [2,3]. In our hands we showed that most Algerian SCC patients were HPV-EBV co-infected.
A lesser degree of co-infection was observed in pre-cancerous lesions of the cervix.
A possible joint effect of the two viruses on cervix tumor development should be considered.
Such hypothesis is strengthened by EBV oncogene expression. It is clear that the presence
of EBV and its relationship with HPV in cervical oncogenesis need to be further investigated.

Materials and methods

Samples

One hundred and nine cervix specimens taken from female patients attending a gynecological
department in Algiers (Algeria) were randomly selected and enrolled for EBV and HPV-DNA
detection and genotyping (Figure 6). We obtained from the Scientific Council of the Faculty of Natural Sciences and
Life, University Setif-1 (Algeria) permission to investigate on human samples, as
required by the Helsinki declaration respecting ethical principles for medical research,
including research on identifiable human samples and data (Ref: CSF/SNV/2013). Patients
were asked to participate in the study, and informed consent was obtained for HPV
testing. They were classified as follows: Group 1 was composed of 58 patients having
invasive or in situ squamous-cell carcinoma (SCC) of the uterine cervix. When the
tumor became evident, biopsies were freshly obtained from these women immediately
after surgery and were snap-frozen in liquid nitrogen, transported to the laboratory
and then stored at −80°C until processed. Histological grading was performed by an
experienced pathologist. Each biopsy was cut with sterile blades, homogenized and
divided into three fractions; used respectively for RNA, DNA, and protein preparations.
Group 2 were 16 patients with low-grade lesions (Cervical intraepithelial neoplasia-1
(CIN-1), condyloma, and cervicitis). Group 3 were 21 women with CIN-2/3 (known as
mild and severe dysplasia). The samples of groups 2 and 3 were collected from the
apparently malignant zone. The samples were then placed into a one ml tube of the
transport medium (ViraPap/Viratype transport medium, (Digene, Silver Spring, MD))
and stored at −20°C until processing. Group 4 were 14 cases of normal cervixes obtained
by scraping the junction area of healthy women undergoing routine cervical screening.
Conventional cytology was performed for this group.

Hybrid Capture 2 (HC2)

The HC2, a standardized US FDA-approved test has been employed to detect one or more
of 13 carcinogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68).
This enzyme-linked immunosorbent assay is based on a sandwich hybridization followed
by a non-radioactive alkaline phosphatase reaction following the manufacturer’s instructions.

Polymerase chain reaction for detection of HPV DNA and β-globin

Specimens were subjected to PCR with general HPV primers using conventional L1 consensus
primers, MY11 (GCMCAGGGWCATAAYAATGG) and MY09 (CGTCCMARRGGAWACTGATC). The assays were
conducted according to the manufacturer’s protocol (Digene SHARP Signal™ System).

EBV-DNA identification by Southern blotting

As previously published by our group [39], EBV PCR was performed in 25 μl reaction mixture containing Taq polymerase buffer
(10 mM Tris–HCl, pH 8.3; 50 mM KCl), 5 mM MgCl2, 200 mM of each deoxyribonucleic triphosphate,
and 250 nM of each primer and 1 microgram of DNA sample. The whole BALF1 sequence
was amplified from the DNA extracts using two primers: ALF1-S (GGGGATCCAATGAACCTGGCCATTGCTCTG)
(upstream) at position 165,517 of EBV genome and ALF1-AS (CGGAATTCTTACAAAGATTTCAGGAAGTC)
(downstream) at position 164,858. These primers permitted to amplify a fragment of
660 bp (the entire BALF-1 gene). Ten microliters of the amplified fragment were loaded
onto 2% agarose gel, electrophoretically separated and revealed by ethidium bromide
and then transferred onto reinforced nitrocellulose membranes (Schleicher & Schuell,
Germany). Amplified fragment was detected by hybridization using 32P-labeled-BamH1-A probe prepared with a random primer DNA-labeling kit (Stratagene)
[33]. The hybridization was carried out in a modified solution described by Ausubel et
al. [34] with 106 cpm/ml of the labeled BALF1 probe.

RT-PCR

RNA was extracted from control and specimens with Trizol reagent (Invitrogen) according
to the manufacturer’s instruction. Total RNA was treated with DNase (amplification
grade Deoxyribonuclease I, Invitrogen). Reverse transcriptase PCR (RT-PCR) was carried
out as previously described [10]. LMP-1 was amplified with primers 5′-CGGGATCCATGGAACGCGACCTTGAGAG and 5′-CGGGATCCCAACAGAAGAGACCTTCTCT.

Immunohistochemical staining

Cervix tissues were formalin fixed, dehydrated in alcohol and embedded in paraffin.
Immunostaining was performed according to the streptavidin-biotin peroxidase complex
method, using monoclonal antibodies against HPV, LMP-1 and EBNA-1. Briefly, sections
were cut to a thickness of 4 μm, mounted on silane-treated slides (Superfrost), and
dried 1 hour at 60°C. All sections were then deparaffinized in xylene, rehydrated
through alcohol, and washed in phosphate-buffered saline. This buffer was used for
all subsequent washes. Sections for LMP-1, EBNA-1 and HPV detection were heated in
a microwave oven twice for 5 min in citrate buffer (pH 6.0). Mouse monoclonal antibodies
(at a dilution of 1:500) anti-LMP-1 (S12, BD Biosciences Pharmingen), and anti-EBNA-1
(sc-29; Santa Cruz; Germany) were used respectively as the primary antibody and incubated
overnight at room temperature followed by a conventional streptavidin peroxidase method
(DAKO, Denmark). Signals were developed with 3, 30-diaminobenzidine for 5 minutes
and counter-stained with hematoxylin. The results were scored for the positive signals
in tissue cells. The same samples were sectioned at 4 μm thickness and routine hematoxylin-
and eosine-stained for histologic examination.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AK for HPV classification and HPV detection, RT-PCR, PCR, Immunohistochemistry, Southern
blotting, immunoblotting, analysis of results and redaction. NS for HPV classification
and HPV detection. AB for preparation of cell scrapings and biopsies of the cervix.
KH for analysis of results. AG for preparation of cell scrapings and biopsies of the
cervix. TO for analysis of results and for redaction. AB for analysis of results and
for redaction. All authors read and approved the final manuscript.

Acknowledgements

This work was financially supported by a grant from ATRSS (Agence Thématique de Recherche
en Sciences de la Santé; grant code : 03/05/01/10/024 for A. Khenchouche), by a grant
from “Agence National de la Recherche MIME programme for T. Ooka and by a grant from
“Cooperation Inter-universitaire Franco-Algerienne” no. 05 MDU 663 for T. Ooka and
A. Bouguermouh. The authors are grateful to Dr. J. Trouillas, Faculty of Medicine
Laennec, University of Lyon-1 for the histological and immunohistochemistry technical
assistance. We also thank M. Benboubetra of the Setif-1 University for critical reading
and English correction of the text.

References

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